Sterol regulatory element binding protein (SREBP-1c) is the primary transcription factor that governs expression of a host of lipogenic genes in the liver. In states of obesity and hyperinsulinemia, SREBP-1c production is increased due, in part, to elevated levels of transcription. First, an animal model of obesity and hyperinsulinemia, the JCR:LA-cp rat, will be employed to determine the effect of dietary supplementation with menhaden oil on the in vivo associations of known transcription factors and coactivators (LXRa, NF-Y, Sp-1, SREBP-1c, SRC-1, CBP/p300) and possible repressers (Kid-1 and NrOb2) with the promoter region of SREBP-1c and its target genes by chromatin immunoprecipitation assays. The effect of menhaden oil supplementation on abundance of these factors will be determined by real-time PCR and western blotting. In addition, novel PUFA regulatory mechanisms will be examined such as transcriptional repression by NrOb2, Kid-1, and phosphorylation of insulin-sensitive transcription factors/coactivators. Secondly, to determine the molecular mechanism(s) by which PUFA repress insulin stimulated SREBP-1c transcription, mutational analysis of transcription factor binding elements within the rat SREBP-1c promoter will be performed to dissect the elements which mediate PUFA effects. Rat SREBP-1c promoter driven luciferase assays in rat primary hepatocytes will be used to determine transcriptional activity. Gene knockdown by siRNA directed against target proteins will be used to confirm promoter mutation studies and plasmid mediated overexpression of candidate transactivators will be used to "rescue" transcriptional activity in the face of PUFAs. Taken together, the presently proposed studies should provide much needed insight into the molecular mechanism(s) through which dietary PUFAs can ameliorate elevations in SREBP-lc expression and the resulting dyslipidemia observed in obesity and hyperinsulinemia. Relevance: The current proposal seeks to determine the mechanism(s) by which long chain polyunsaturated fatty acids (PUFAs) decrease expression of SREBP-1c and lipogenesis in the obese state thereby ameliorating dyslipidemia associated with obesity and type II diabetes. [unreadable] [unreadable] [unreadable]